The enzyme D-amino acid oxidase is known to be useful in tests to detect the presence of D-alanine. One such assay of particular commercial importance is used to detect the presence of beta-lactam antibiotics in milk. Assays for beta-lactam antibiotics (e.g., penicillins and cephalosporins) are important because large quantities of milk must be discarded each year due to antibiotic contamination.
Frere et al ("Enzymatic Method for Rapid and Sensitive Determination of beta-lactam Antibiotics" Antimicrobial Agents and Chemotherapy, Oct. 1980 pp. 506-510) describe an enzymatic assay for beta-lactam antibiotics using D-amino acid oxidase as part of a reagent system which produces a color change in the presence of D-alanine. That assay relies on the ability of beta-lactam antibiotics to inactivate a specific D,D-carboxypeptidase produced by Actinomadura R39. Other streptomyces D,D-carboxypeptidases are known to be reversably inhibited by beta-lactam antibiotics, but the R39 enzyme is preferred because the rate of inactivation is very rapid and the reversal of inhibition is very slow.
The assay as described by Frere et al is similar to a commercial test known as "Penzym".RTM. sold by UCB Bioproducts, Brussels, Belgium. The Penzym.RTM. system is sold in seven vials and involves seven steps which take an extended period (20 to 30 minutes).
In the first step D,D-carboxypeptidase is added to the sample and incubated for a period of time, e.g., five minutes. If the sample contains beta-lactam antibiotic, the enzyme will be inactivated to a degree depending on the amount of antibiotic present. Next a substrate for D,D-carboxypeptidase which is a peptide containing a carboxyterminal D-alanine is added.
After further incubation (e.g., 15 minutes), other reagents are added to detect liberation of D-alanine from the substrate. These reagents include D-amino acid oxidase, its co-factor flavin adenine dinucleotide, a peroxide sensitive dye and peroxidase to catalyze the color forming reaction. D-amino acid oxidase oxidizes the D-alanine into pyruvic acid with simultaneous formation of hydrogen peroxide. The hydrogen peroxide oxidizes an organic redox indicator, e.g, o-dianisidine, which produces a distinctive color. At the end of the incubation period a strong acid is added to terminate the reaction and stabilize the color formation.
The Penzym.RTM. kit is supplied with seven separate reagents including: (1) the D,D-carboxypeptidase; (2) a buffer for the D,D-carboxypeptidase; (3) substrate for the D,D-carboxypeptidase, N,N,Diacetyl-2-L-lysyl-D-alanyl-D-alanine: (4) flavin adenine dinucleotide, a cofactor of the D-amino acid oxidase; (5) peroxidase; (6) ortho-dianisidine; and (7) D-amino acid oxidase. The Penzym.RTM. assay suffers several disadvantages which make it less than acceptable to dairy farmers and milk haulers. Among the most significant disadvantages is the requirement for handling an excessive number of separately packaged aqueous reagents.
Many researchers have suggested impregnating paper and other materials with reagents which can be conveniently used for "dip-and-read" tests. For example, Johnson et al, U.S. Pat. No. 4,046,514, September 6, 1977, teach impregnating fibers with reagents and thereafter forming the fibers into a carrier matrix. The examples include a self-calibrated glucose test incorporating glucose oxidase in cotton fibers which are woven into the test strip. Bauer, U.S. Pat. No. 4,390,621, June 28, 1983, suggests impregnating filter paper with glucose oxidase and peroxidase. Lange et al, U.S. Pat. No. 3,802,842, April 9, 1974, suggest covering a filter paper test strip impregnated with reagents for detection of glucose, including glucose oxidase and peroxidase, with a fine fabric meshwork including polyester fabric and nylon fabric.
Enzymes are well known for their lack of predictability in terms of stability. Thus, while glucose oxidase and peroxidase retain their activity when dried on cellulosic-based materials D-amino acid oxidase has been found to lose its activity when dried on cellulosic materials.